Resistance to demethylation inhibitors in Cercospora beticola, a pathogen of sugar beet in Japan, and development of unique cross-resistance patterns

Cercospora beticola Sacc. is the most destructive pathogen of sugar beet (Beta vulgaris L.) and causes Cercospora leaf spot (CLS). Since 1986, fungicides that function as demethylation inhibitors (DMIs) have been used to control CLS in Hokkaido, which is the only area in Japan where sugar beet is grown. Reduced sensitivity of C. beticola to DMI fungicides, based on the half maximal effective concentration (EC50), was first reported in Hokkaido in 1999, however the fungicides continued to be used effectively until 2014. In a field experiment in 2016, we found that the efficacy of difenoconazole against the field population of C. beticola was greatly reduced. We subsequently tested over 600 isolates collected throughout the sugar beet-growing region of Hokkaido and revealed that the mean resistance factor of four DMI fungicides (difenoconazole, fenbuconazole, tebuconazole, and tetraconazole) were high, which indicates that DMI-resistant isolates were distributed throughout the beet cultivation area. Moreover, we identified three types of isolates that have unique cross-resistance patterns between difenoconazole and fenbuconazole, with their EC50 rate (= difenoconazole EC50/ fenbuconazole EC50) converged to 31, 4.0, and 0.40, respectively, which appeared to be affected by the local history of fungicide usage. The F144L substitution in CbCYP51 was only found in the group whose EC50 rate was 0.40. This is the first report of DMI resistance in C. beticola in Japan, and the findings in this study could contribute to our understanding of the mechanism of DMI resistance.